Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells

BACKGROUND Liver fibrosis is the common pathological process associated with the occurrence and development of various chronic liver diseases.At present,there is still a lack of effective prevention and treatment methods in clinical practice.Hepatic stellate cell(HSC)plays a key role in liver fibrogenesis.In recent years,the study of liver fibrosis targeting HSC autophagy has become a hot spot in this research field.Angiotensin-converting enzyme 2(ACE2)is a key negative regulator of reninangiotensin system,and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored.AIM To investigate the effect of ACE2 on hepatic fibrosis in mice by regulating HSC autophagy through the Adenosine monophosphate activates protein kinases(AMPK)/mammalian target of rapamycin(mTOR)pathway.METHODS Overexpression of ACE2 in a mouse liver fibrosis model was induced by injection of liver-specific recombinant adeno-associated virus ACE2 vector(rAAV2/8-ACE2).The degree of liver fibrosis was assessed by histopathological staining and the biomarkers in mouse serum were measured by Luminex multifactor analysis.The number of apoptotic HSCs was assessed by terminal deoxynucleoitidyl transferase-mediated dUTP nick-end labeling(TUNEL)and immunofluorescence staining.Transmission electron microscopy was used to identify the changes in the number of HSC autophagosomes.The effect of ACE2 overexpression on Wu Y et al.ACE2 improves liver fibrosis through autophagy WJG https://www.wjgnet.com 4976 September 7,2023 Volume 29 Issue 33 autophagy-related proteins was evaluated by multicolor immunofluorescence staining.The expression of autophagy-related indicators and AMPK pathway-related proteins was measured by western blotting.RESULTS A mouse model of liver fibrosis was successfully established after 8 wk of intraperitoneal injection of carbon tetrachloride(CCl4).rAAV2/8-ACE2 administration reduced collagen deposition and alleviated the degree of liver fibrosis in mice.The serum levels of platelet-derived growth factor,angiopoietin-2,vascular endothelial growth factor and angiotensin II were decreased,while the levels of interleukin(IL)-10 and angiotensin-(1-7)were increased in the rAAV2/8-ACE2 group.In addition,the expression of alpha-smooth muscle actin,fibronectin,and CD31 was down-regulated in the rAAV2/8-ACE2 group.TUNEL and immunofluorescence staining showed that rAAV2/8-ACE2 injection increased HSC apoptosis.Moreover,rAAV2/8-ACE2 injection notably decreased the number of autophagosomes and the expression of autophagy-related proteins(LC3I,LC3II,Beclin-1),and affected the expression of AMPK pathway-related proteins(AMPK,p-AMPK,p-mTOR).CONCLUSION ACE2 overexpression can inhibit HSC activation and promote cell apoptosis by regulating HSC autophagy through the AMPK/mTOR pathway,thereby alleviating liver fibrosis and hepatic sinusoidal remodeling.

Hydroxysafflor yellow A protects against thioacetamide-induced liver fibrosis in rats via suppressing proinflammatory/fibrogenic mediators and promoting hepatic stellate cell senescence and apoptosis

Objective:To evaluate the effect of hydroxysafflor yellow A(HSYA)on thioacetamide-induced liver fibrosis.Methods:Thioacetamide was administered to rats intraperitoneally in doses of 200 mg/kg twice a week for 12 weeks.Thioacetamide-intoxicated rats were given silymarin(50 mg/kg)or HSYA(5 mg/kg)orally every day for 8 weeks.Liver enzymes,fibrosis markers,histological changes as well as immunohistochemistry of TNF-α,IL-6,p21,α-SMA,and caspase-3 were examined.The effect of HSYA on HSC-T6 activation/proliferation and apoptosis was also determined in vitro.Results:HSYA decreased liver enzymes,TNF-α,IL-6,and p21 expressions,hepatic PDGF-B,TIMP-1,TGF-β1,and hydroxyproline levels,as well as fibrosis score(S2 vs.S4)compared to the thioacetamide group.HSYA also downregulatedα-SMA while increasing caspase-3 expression.Surprisingly,at 500μg/mL,HSYA had only a slightly suppressive effect on HSC proliferation,with a 9.5%reduction.However,it significantly reduced TGF-β1,inhibitedα-SMA expression,induced caspase-3 expression,and promoted cell senescence.Conclusions:HSYA may be a potential therapeutic agent for delaying and reversing the progression of liver fibrosis.More research on HSYA at higher doses and for a longer period is warranted.

Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress

BACKGROUND Cryptotanshinone(CPT)has wide biological functions,including anti-oxidative,antifibrosis,and anti-inflammatory properties.However,the effect of CPT on hepatic fibrosis is unknown.AIM To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action.METHODS Hepatic stellate cells(HSCs)and normal hepatocytes were treated with different concentrations of CPT and salubrinal.The CCK-8 assay was used to determine cell viability.Flow cytometry was used to measure apoptosis and cell cycle arrest.Reverse transcription polymerase chain reaction(RT-PCR)and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress(ERS)signaling pathway related molecules,respectively.Carbon tetrachloride(CCL4)was used to induce in vivo hepatic fibrosis in mice.Mice were treated with CPT and salubrinal,and blood and liver samples were collected for histopathological examination.RESULTS We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro.CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs.Furthermore,we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers(CHOP and GRP78)and activating ERS pathway molecules(PERK,IRE1α,and ATF4),which were inhibited by salubrinal.Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model.CONCLUSION CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway,which represents a promising strategy for treating hepatic fibrosis.

BMI-1 activates hepatic stellate cells to promote the epithelialmesenchymal transition of colorectal cancer cells

BACKGROUND Activated hepatic stellate cells(aHSCs)are the major source of cancer-associated fibroblasts in the liver.Although the crosstalk between aHSCs and colorectal cancer(CRC)cells supports liver metastasis(LM),the mechanisms are largely unknown.AIM To explore the role of BMI-1,a polycomb group protein family member,which is highly expressed in LM,and the interaction between aHSCs and CRC cells in promoting CRC liver metastasis(CRLM).METHODS Immunohistochemistry was carried out to examine BMI-1 expression in LM and matched liver specimens of CRC.The expression levels of BMI-1 in mouse liver during CRLM(0,7,14,21,and 28 d)were detected by Western blotting(WB)and the quantitative polymerase chain reaction(qPCR)assay.We overexpressed BMI-1 in HSCs(LX2)by lentivirus infection and tested the molecular markers of aHSCs by WB,qPCR,and the immunofluorescence assay.CRC cells(HCT116 and DLD1)were cultured in HSC-conditioned medium(LX2 NC CM or LX2 BMI-1 CM).CM-induced CRC cell proliferation,migration,epithelial-mesenchymal transition(EMT)phenotype,and transforming growth factor beta(TGF-β)/SMAD pathway changes were investigated in vitro.A mouse subcutaneous xenotransplantation tumor model was established by co-implantation of HSCs(LX2 NC or LX2 BMI-1)and CRC cells to investigate the effects of HSCs on tumor growth and the EMT phenotype in vivo.RESULTS Positive of BMI-1 expression in the liver of CRLM patients was 77.8%.The expression level of BMI-1 continued to increase during CRLM in mouse liver cells.LX2 overexpressed BMI-1 was activated,accompanied by increased expression level of alpha smooth muscle actin,fibronectin,TGF-β1,matrix metalloproteinases,and interleukin 6.CRC cells cultured in BMI-1 CM exhibited enhanced proliferation and migration ability,EMT phenotype and activation of the TGF-β/SMAD pathway.In addition,the TGF-βR inhibitor SB-505124 diminished the effect of BMI-1 CM on SMAD2/3 phosphorylation in CRC cells.Furthermore,BMI-1 overexpressed LX2 HSCs promoted tumor growth and the EMT phenotype in vivo.CONCLUSION High expression of BMI-1 in liver cells is associated with CRLM progression.BMI-1 activates HSCs to secrete factors to form a prometastatic environment in the liver,and aHSCs promote proliferation,migration,and the EMT in CRC cells partially through the TGF-β/SMAD pathway.

Mechanism of annexin A1/N-formylpeptide receptor regulation of macrophage function to inhibit hepatic stellate cell activation through Wnt/β-catenin pathway

BACKGROUND Hepatic fibrosis is a common pathological process of chronic liver diseases with various causes,which can progress to cirrhosis.AIM To evaluate the effect and mechanism of action annexin(Anx)A1 in liver fibrosis and how this could be targeted therapeutically.METHODS CCl4(20%)and active N-terminal peptide of AnxA1(Ac2-26)and N-formylpeptide receptor antagonist N-Boc-Phe-Leu-Phe-Leu-Phe(Boc2)were injected intraperitoneally to induce liver fibrosis in eight wild-type mice/Anxa1 knockout mice,and to detect expression of inflammatory factors,collagen deposition,and the role of the Wnt/β-catenin pathway in hepatic fibrosis.RESULTS Compared with the control group,AnxA1,transforming growth factor(TGF)-β1,interleukin(IL)-1βand IL-6 expression in the liver of mice with hepatic fibrosis induced by CCl4 was significantly increased,which promoted collagen deposition and expression ofα-smooth muscle actin(α-SMA),collagen type I and connective tissue growth factor(CTGF),and increased progressively with time.CCl4 induced an increase in TGF-β1,IL-1βand IL-6 in liver tissue of AnxA1 knockout mice,and the degree of liver inflammation and fibrosis and expression ofα-SMA,collagen I and CTGF were significantly increased compared with in wild-type mice.After treatment with Ac2-26,expression of liver inflammatory factors,degree of collagen deposition and expression of a-SMA,collagen I and CTGF were decreased compared with before treatment.Boc2 inhibited the anti-inflammatory and antifibrotic effects of Ac2-26.AnxA1 downregulated expression of the Wnt/β-catenin pathway in CCl4-induced hepatic fibrosis.In vitro,lipopolysaccharide(LPS)induced hepatocyte and hepatic stellate cell(HSC)expression of AnxA1.Ac2-26 inhibited LPS-induced RAW264.7 cell activation and HSC proliferation,decreased expression ofα-SMA,collagen I and CTGF in HSCs,and inhibited expression of the Wnt/β-catenin pathway after HSC activation.These therapeutic effects were inhibited by Boc2.CONCLUSION AnxA1 inhibited liver fibrosis in mice,and its mechanism may be related to inhibition of HSC Wnt/β-catenin pathway activation by targeting formylpeptide receptors to regulate macrophage function.

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background:Activated hepatic stellate cells(HSCs)are closely involved in the initiation,perpetuation,and resolution of liver fibrosis.Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis.Methods:In this study,we investigated the effect of the pro-inflammatory cytokine interleukin(IL)-1βon the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells,an HSC cell line,using western blotting and cell proliferation assays.Results:IL-1βincreased the proliferation rate andα-smooth muscle actin(SMA)expression of LX-2 cells in a dose-dependent manner.Within 1 h after IL-1βtreatment,c-Jun N-terminal kinase(JNK),p38,and nuclear factor-κB(NF-κB)signaling was activated in LX-2 cells.Subsequently,protein kinase B(AKT)phosphorylation and an increase inα-SMA expression were observed in LX-2 cells.Each inhibitor of JNK,p38,or NF-κB decreased cell proliferation,AKT phosphorylation,andα-SMA expression in IL-1β-treated LX-2 cells.Conclusion:These results indicate that JNK,p38,and NF-κB signals converge at AKT phosphorylation,leading to LX-2 activation by IL-1β.Therefore,the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.

Daily genetic profiling indicates JAK/STAT signaling promotes early hepatic stellate cell transdifferentiation

AIM: To identify signaling pathways and genes that initiate and commit hepatic stellate cells (HSCs) to transdifferentiation. METHODS: Primary HSCs were isolated from male Sprague-Dawley rats and cultured on plastic for 0-10 d. Gene expression was assessed daily (quiescent to day 10 culture-activation) by real time polymerase chain reaction and data clustered using AMADA software. The significance of JAK/STAT signaling to HSC transdifferentiation was determined by treating cells with a JAK2 inhibitor. RESULTS: Genetic cluster analyses, based on expression of these 21 genes, showed similar expression profiles on days 1-3, days 5 and 6, and days 7-10, while freshly isolated cells (day Q) and day 4 cells were genotypically distinct from any of the other days. Additionally, gene expression clustering revealed strong upregulation of interleukin-6, JAK2 and STAT3 mRNA in the early stages of activation. Inhibition of the JAK/STAT signaling pathway impeded the morphological transdifferentiation of HSCs which correlated with decreased mRNA expression of several profibrotic genes including collagens, α-SMA, PDGFR and TGFβR. CONCLUSION: These data demonstrate unique clustered genetic profiles during the daily progression of HSC transdifferentiation and that JAK/STAT signaling may be critical in the early stages of transdifferentiation.

Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

AIM:To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultraperform ance liquid chromatographyelectrospray ionizationtandem mass spectrometry (UPLCESIMS/MS) was used to study the differential expression of proteins and Western blotting was used to validate the results. Gene ontology (GO) method was utilized to analyze the functional enrichment of differentially expressed proteins. Flow cytometry was performed to compare the apoptosis rate between taurinetreated and untreated hepatic stellate cells (HSCs).RESULTS: Nineteen differentially expressed proteins (11 upregulated and 8 downregulated) were identifiedby 2D/MS, and the expression profiles of GLO1 and ANXA1 were validated by Western blotting. GO analysis found that these differentially expressed proteins were enriched within biological processes such as "cellular apoptosis", "oxidation reaction" and "metabolic process" in clusters. Flow cytometric analysis showed that taurinetreated HSCs had a significantly increased apoptosis rate when compared with the control group.CONCLUSION: Natural taurine can promote HSC apoptosis so as to inhibit hepatic fibrosis.

Anti-proliferative and pro-apoptotic effects of tectorigenin on hepatic stellate cells

AIM: To investigate the effect of tectorigenin on proliferation and apoptosis of hepatic stellate cells (HSC)-T6 cells. METHODS: HSC-T6 cells were incubated with tectorigenin at different concentrations, and their proliferation was assessed by bromodeoxyuridine incorporation assay. Apoptosis was detected by flow cytometry assay with Hoechst 33342 staining. Also, generation of reactive oxygen species (ROS), intracellular [Ca2+]i, potential of mitochondrial membrane, activities of cytochrome c and caspase-9 and-3 were investigated to explore a conceivable apoptotic pathway. RESULTS: Tectorigenin suppressed the proliferation of HSC-T6 cells and induced apoptosis of HSC-T6 cells in a time-and dose-dependent manner. Tectorigenin at the concentration of 100 μg/mL greatly inhibited the viability of HSC-T6 cells and induced the condensation of chromatin and fragmentation of nuclei. When treated for 48 h, the percentage of cell growth and apoptosis reached 46.3% ± 2.37% (P = 0.004) and 50.67% ± 3.24% (P = 0.003), respectively. Furthermore, tectorigenin-induced apoptosis of HSC-T6 cells was associated with the generation of ROS, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3. CONCLUSION: Tectorigenin inhibits proliferation of HSC-T6 cells and induces apoptosis of HSC-T6 cells.

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of collagen metabolism proteins,including matrix metalloproteinases-13(MMP-13) and tissue inhibitors of metalloproteinases-1(TIMP-1) was also determined by both real-time Q-PCR and Western blotting analysis.RESULTS:The transfection of FAK shRNA plasmids into HSC resulted in disrupted FAK expression.Compared with the HK group,the levels of type collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif icantly decreased(0.69 ± 0.03 vs 1.96 ± 0.15,P = 0.000;0.59 ± 0.07 vs 1.62 ± 0.12,P = 0.020).The production of TIMP-1 in this cell type was also signif icantly reduced at both mRNA and protein levels(0.49 ± 0.02 vs 1.72 ± 0.10,P = 0.005;0.76 ± 0.08 vs 2.31 ± 0.24,P = 0.000).However,the expression of MMP-13 mRNA could be significantly up-regulated by the transfection of FAK shRNA plasmids into HSC(1.74 ± 0.20 vs 1.09 ± 0.09,P = 0.000).CONCLUSION:These data support the hypothesis that shRNA-mediated disruption of FAK expression could attenuate extracellular matrix(ECM) synthesis and promote ECM degradation,making FAK a potential target for novel anti-f ibrosis therapies.

Mitochondria: A critical hub for hepatic stellate cells activation during chronic liver diseases

Background: Upon liver injury, quiescent hepatic stellate cells(q HSCs), reside in the perisinusoidal space, phenotypically transdifferentiate into myofibroblast-like cells(MFBs). The q HSCs in the normal liver are less fibrogenic, migratory, and also have less proliferative potential. However, activated HSCs(a HSCs) are more fibrogenic and have a high migratory and proliferative MFBs phenotype. HSCs activation is a highly energetic process that needs abundant intracellular energy in the form of adenosine triphosphate(ATP) for the synthesis of extracellular matrix(ECM) in the injured liver to substantiate the injury. Data sources: The articles were collected through Pub Med and EMBASE using search terms "mitochondria and hepatic stellate cells", "mitochondria and HSCs", "mitochondria and hepatic fibrosis", "mitochondria and liver diseases", and "mitochondria and chronic liver disease", and relevant publications published before September 31, 2020 were included in this review. Results: Mitochondria homeostasis is affected during HSCs activation. Mitochondria in a HSCs are highly energetic and are in a high metabolically active state exhibiting increased activity such as glycolysis and respiration. a HSCs have high glycolytic enzymes expression and glycolytic activity induced by Hedgehog(Hh) signaling from injured hepatocytes. Increased glycolysis and aerobic glycolysis(Warburg effect) endproducts in a HSCs consequently activate the ECM-related gene expressions. Increased Hh signaling from injured hepatocytes downregulates peroxisome proliferator-activated receptor-γ expression and decreases lipogenesis in a HSCs. Glutaminolysis and tricarboxylic acid cycle liberate ATPs that fuel HSCs to proliferate and produce ECM during their activation. Conclusions: Available studies suggest that mitochondria functions can increase in parallel with HSCs activation. Therefore, mitochondrial modulators should be tested in an elaborate manner to control or prevent the HSCs activation during liver injury to subsequently regress hepatic fibrosis.

Pathophysiological mechanisms of hepatic stellate cells activation in liver fibrosis

Liver fibrosis is a complex pathological process controlled by a variety of cells,mediators and signaling pathways.Hepatic stellate cells play a central role in the development of liver fibrosis.In chronic liver disease,hepatic stellate cells undergo dramatic phenotypic activation and acquire fibrogenic properties.This review focuses on the pathophysiological mechanisms of hepatic stellate cells activation in liver fibrosis.They enter the cell cycle under the influence of various triggers.The“Initiation”phase of hepatic stellate cells activation overlaps and continues with the“Perpetuation”phase,which is characterized by a pronounced inflammatory and fibrogenic reaction.This is followed by a resolution phase if the injury subsides.Knowledge of these pathophysiological mechanisms paved the way for drugs aimed at preventing the development and progression of liver fibrosis.In this respect,impairments in intracellular signaling,epigenetic changes and cellular stress response can be the targets of therapy where the goal is to deactivate hepatic stellate cells.Potential antifibrotic therapy may focus on inducing hepatic stellate cells to return to an inactive state through cellular aging,apoptosis,and/or clearance by immune cells,and serve as potential antifibrotic therapy.It is especially important to prevent the formation of liver cirrhosis since the only radical approach to its treatment is liver transplantation which can be performed in only a limited number of countries.

Lipotoxic hepatocyte-derived exosomal miR-1297 promotes hepatic stellate cell activation through the PTEN signaling pathway in metabolic-associated fatty liver disease

BACKGROUND Exosomes play an important role in metabolic-associated fatty liver disease(MAFLD),but the mechanism by which exosomes participate in MAFLD still remain unclear.AIM To figure out the function of lipotoxic exosomal miR-1297 in MAFLD.METHODS MicroRNA sequencing was used to detect differentially expressed miRNAs(DEmiR)in lipotoxic exosomes derived from primary hepatocytes.Bioinformatic tools were applied to analyze the target genes and pathways regulated by the DE-miRs.Quantitative real-time PCR(qPCR)was conducted for the verification of DEmiRs.qPCR,western blot,immunofluorescence staining and ethynyl-20-deoxyuridine assay were used to evaluate the function of lipotoxic exosomal miR-1297 on hepatic stellate cells(LX2 cells).A luciferase reporter experiment was performed to confirm the relationship of miR-1297 and its target gene PTEN.RESULTS MicroRNA sequencing revealed that there were 61 exosomal DE-miRs(P<0.05)with a fold-change>2 from palmitic acid treated primary hepatocytes compared with the vehicle control group.miR-1297 was the most highly upregulated according to the microRNA sequencing.Bioinformatic tools showed a variety of target genes and pathways regulated by these DE-miRs were related to liver fibrosis.miR-1297 was overexpressed in exosomes derived from lipotoxic hepatocytes by qPCR.Fibrosis promoting genes(α-SMA,PCNA)were altered in LX2 cells after miR-1297 overexpression or miR-1297-rich lipotoxic exosome incubation via qPCR and western blot analysis.Immunofluorescence staining and ethynyl-20-deoxyuridine staining demonstrated that the activation and proliferation of LX2 cells were also promoted after the above treatment.PTEN was found to be the target gene of miR-1297 and knocking down PTEN contributed to the activation and proliferation of LX2 cells via modulating the PI3K/AKT signaling pathway.CONCLUSION miR-1297 was overexpressed in exosomes derived from lipotoxic hepatocytes.The lipotoxic hepatocyte-derived exosomal miR-1297 could promote the activation and proliferation of hepatic stellate cells through the PTEN/PI3K/AKT signaling pathway,accelerating the progression of MAFLD.

Metabolomic profiling for identification of metabolites and relevant pathways for taurine in hepatic stellate cells

AIM To develop a reliable and simple method to identify important biological metabolites and relevant pathways for taurine in hepatic stellate cells(HSCs), in order to provide more data for taurine therapy.METHODS All the biological samples were analyzed by using highperformance liquid chromatography-time electrospray ionization/quadrupole-time of flight mass spectrometry. Principal component analysis and partial least squares discriminant analysis were used to identify statistically different metabolites for taurine in HSCs, and metabolomic pathway analysis was used to do pathway analysis for taurine in HSCs. The chemical structure of the related metabolites and pathways was identified by comparing the m/z ratio and ion mode with the data obtained from free online databases.RESULTS A total of 32 significant differential endogenous metabolites were identified, which may be related to the mechanism of action of taurine in HSCs. Among the seven relevant pathways identified, sphingolipid metabolism pathway, glutathione metabolism pathway and thiamine metabolism pathway were found to be the most important metabolic pathways for taurine in HSCs.CONCLUSION This study showed that there were distinct changes in biological metabolites of taurine in HSCs and three differential metabolic pathways including sphingolipid pathway, glutathione pathway and thiamine metabolism pathway might be of key importance in mediating the mechanism of action of taurine in HSCs.

Rosmarinic acid attenuates hepatic fibrogenesis via suppression of hepatic stellate cell activation/proliferation and induction of apoptosis

Objective:To investigate the antifibrotic role of rosmarinic acid(RA),a natural polyphenolic compound,on HSCs activation/proliferation and apoptosis in vitro and in vivo. Methods:The impact of RA on stellate cell line(HSC-T6) proliferation,activation and apoptosis was assessed along with its safety on primary hepatocytes. In vivo,rats were divided into:(i) normal;(ii) thioacetamide(TAA)-intoxicated rats for 12 weeks;(iii) TAA+silymarin or(iv) TAA+RA. At the end of experiment,liver functions,oxidative stress,inflammatory and profibrogenic markers,tissue inhibitor metalloproteinases type-1(TIMP-1) and hydroxyproline(HP) levels were evaluated. Additionally,liver histopathology and immunohistochemical examinations of alpha-smooth muscle actin(α-SMA),caspase-3 and proliferation cellular nuclear antigen(PCNA) were determined. Results:RA exhibited anti-proliferative effects on cultured HSCs in a time and concentration dependent manner showing an IC50 of 276 μg/mL and 171 μg/mL for 24 h and 48 h,respectively,with morphological reversion of activated stellate cell morphology to quiescent form. It significantly improved ALT,AST,oxidative stress markers and reduced TIMP-1,HP levels,inflammatory markers and fibrosis score(S1 vs S4). Furthermore,reduction in α-SMA plus elevation in caspase-3 expressions of HSCs in vitro and in vivo associated with an inhibition in proliferation of damaged hepatocytes were recorded. Conclusions:RA impeded the progression of liver fibrosis through inhibition of HSCs activation/proliferation and induction of apoptosis with preservation of hepatic architecture.

Studies on the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells

OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.

Cross-talk between hepatic stellate cells and T lymphocytes in liver fibrosis

Background: Fibrosis results from inflammation and healing following injury. The imbalance between extracellular matrix(ECM) secretion and degradation leads to the ECM accumulation and liver fibrosis. This process is regulated by immune cells. T lymphocytes, including alpha beta( αβ) T cells, which have adaptive immune functions, and gamma delta( γδ) T cells, which have innate immune functions, are considered regulators of liver fibrosis. This review aimed to present the current understanding of the cross-talk between T lymphocytes and hepatic stellate cells(HSCs), which are the key cells in liver fibrosis. Data sources: The keywords "liver fibrosis", "immune", and "T cells" were used to retrieve articles published in Pub Med database before January 31, 2020. Results: The ratio of CD8 +(suppressor) T cells to CD4+(helper) T cells is significantly higher in the liver than in the peripheral blood. T cells secrete a series of cytokines and chemokines to regulate the inflammation in the liver and the activation of HSCs to influence the course of liver fibrosis. In addition, HSCs also regulate the differentiation and proliferation of T cells. Conclusions: The cross-talk between T cells and HSCs regulates liver fibrosis progression. The elucidation of this communication process will help us to understand the pathological process of liver fibrosis.

Effect of Kruppel-like Factor 4 on Notch Pathway in Hepatic Stellate Cells

The relationship between Kruppel-like factor 4(KLF4) and the Notch pathway was determined to investigate the effect of KLF4 on the activation of hepatic stellate cells and underlying mechanisms.Fifty SPF BALB/c mice were randomly divided into two groups.A liver fibrosis model was established in 25 mice as the experimental group,and the remaining 25 mice served as controls.On the day 0,7,14,and 35,liver tissues were removed for immunofluorescent detection.The Notch pathway inhibitor DAPT was added to the primary original hepatic stellate cells,and KLF4 and Notch-associated factor expression was detected by q RT-PCR.Additionally,the hepatic stellate cell line LX-2 was used to establish control and experimental groups,and was cultured in vitro.LX-2 cells in the experimental groups were treated with DAPT and the Notch activator transforming growth factor-beta 1 separately,whereas those in the control group were given isotonic culture medium.After 48 h,KLF4 expression was examined by Western blotting.After transient transfection of LX-2 cells to increase KLF4,the expression of Notch factor was examined.Immunofluorescence analysis showed that,with the aggravation of liver fibrosis,the absorbance(A) values of KLF4 were decreased(day 0:980.73±153.19;day 7:1087.99±230.23;day 14:390.95±93.56;day 35:245.99±87.34).The expression of Notch pathway-related factors(Notch-1,Notch-2,and Jagged-1) in the hepatic stellate cell membrane was negatively correlated to KLF4 expression.With the increase of KLF4 expression,Notch-2(0.73±0.13) and Jagged-1(0.43±0.12) expression decreased,whereas Notch-1 level was not detectable.When the Notch pathway was inhibited,KLF4 levels generally increased(18.12±1.31).Our results indicate that KLF4 expression is negatively correlated to the Notch pathway in hepatic stellate cells,which may provide a reference for the treatment of hepatic fibrosis.

Naringenin prevented nonalcoholic steatohepatitis fibrosis via regulating MAPK/FoxO3a pathway and promoting apoptosis of activated hepatic stellate cells

OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug treatment could reverse early liver fibrosis and is of significance to prevent NASH from progressing into cirrhosis and liver cancer.Identification of drug targets for NASH treatment has been an active research area and is essential for the development of anti-NASH medications.Naringenin(NGN)is a flavonoid compound rich in citrus fruits.Our preliminary data demonstrated that NGN reduced diet-induced lipid accumulation and inflammation in the mouse liver,but whether NGN can attenuate liver fibrosis of NASH is not known.METHODS To study the effect of NGN on NASH fibrosis.WT mice were fed with high fat diet(HFD)and injected intraperitoneally 20%carbon tetrachloride at the same time for 8 weeks to induce NASH,and NGN was administrated by gavage in the meantime.In vitro,LO2 cells and LX2 cells were stimulated by oleic acid(OA)combined with lipopolysaccharide(LPS),respectively.RESULTS Treating the WT mice with NGN 100 mg·kg^(-1)·d-1 significantly attenuated hepatic lipid accumulation,hepatic fibrosis,plasma ALT and AST levels,inhibited protein expression of p-ERK,p-FoxO3a in the mouse livers.In vitro,on OA and LPS stimulated LO2 or LX2 cells,NGN significantly promoted apoptosis of activated hepatic stellate cells while inhibited apoptosis of hepatocytes.Mechanism study indicated that NGN inhibited MAPK pathway and promoted activation of FoxO3a,consequently promoted apoptosis of the activated LX2 cells and inhibited liver fibrosis.CONCLUSION NGN preventes NASH fibrosis via regulating MAPK/FoxO3a pathway,thus promoting apoptosis of the activated hepatic stellate cells.

Fasudil prevents liver fibrosis via activating natural killer cells and suppressing hepatic stellate cells

BACKGROUND Fasudil,as a Ras homology family member A(RhoA)kinase inhibitor,is used to improve brain microcirculation and promote nerve regeneration clinically.Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis.AIM To evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide(TAA).METHODS C57BL/6 mice were administered TAA once every 3 d for 12 times.At 1 wk after induction with TAA,Fasudil was intraperitoneally injected once a day for 3 wk,followed by hematoxylin and eosin staining,sirius red staining,western blotting,and quantitative polymerase chain reaction(qPCR),and immune cell activation was assayed by fluorescence-activated cell sorting.Furthermore,the effects of Fasudil on hepatic stellate cells and natural killer(NK)cells were assayed in vitro.RESULTS First,we found that TAA-induced liver injury was protected,and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment.Furthermore,western blot and qPCR assays showed that the levels of alpha smooth muscle actin(α-SMA),matrix metalloproteinase 2(MMP-2),MMP-9,and transforming growth factor beta 1(TGF-β1)were inhibited by Fasudil.Moreover,flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro.Furthermore,Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasingα-SMA and TGF-β1.CONCLUSION Fasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation,thereby providing a feasible solution for the clinical treatment of liver fibrosis.